| Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of chronic non-progressive pneumonia in sheep, goat, bighorn and wild small ruminants. Progressive wasting, spasmodic cough and anemia are some of the characteristic symptoms of the disease. The progression of the disease is usually slow that may span from a few months to years. In China, this pathogen was found in Sichuan, Xinjiang, Ningxia, Hubei and other provinces now. It has been a worldwide epidemic that has caused huge economic loss to the sheep industry for decades. Immunological defence and serological detection are still the most effective method to control the disease, so the research of vaccine and serological diagnostic method is necessary. In order to provide the information of antigens for subunit vaccine preparation and diagnosis of M. ovipneumoniae, the immunogenicity of the elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP70) were evaluated in this study. ELISA method using recombinant HSP70 protein as diagnostic antigen was established to detect the anti-M. ovipneumoniae antibody.EF-Tu and HSP70 of M. ovipneumoniae Mo-1 were expressed, screened and purified from Escherichia coli. We found both elongation factor Tu(EF-Tu) and heat shock protein 70 (HSP 70) were the components of TritonX-114 extracted M. ovipenumoniae membrane protein by Western blot with anti-rEF-Tu sera and anti-rHSP70 sera. Immunogold electron micrographs also suggest that both EF-Tu and HSP70 are membrane-associated proteins of M. ovipneumoniea. Humoral and cellular immune responses of the purified recombinant proteins were evaluated by qualitative and quantitative antibody screening methods. Antibody levels, antibody subtypes and cytokines in BALB/c mice were assessed and the results showed increased levels of IgG (contain IgGl and IgG2a), IFN-γ, TNF-a, IL-12(p70), IL-4, IL5 and IL-6 in the sera of both rHSP70 and rEF-Tu treated mice. In addition, ELISPOT assay showed highest increased IFN-γ+ secreting lymphocytes were detected in rHSP70 group. Growth inhibition test (GIT) of Mo showed that sera from rHSP70 and rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins that could induce humoral and cellular immune responses. Therefore, these two proteins may be potential candidates for M. ovipneumoniae vaccine research.We used guinea pig serum complement C3b (extracted with inulin) to select the monoclonal antibody (mAb) of C3b. The mAb B7A7 showed high affinity and specificity for guinea pig C3b a’ chain. According to rHSP70 protein is a conserved protein in M. ovipneumoniae and could induce a better immune response in mice, we used rHSP70 as the coating antigen to establish a Mo-rHSP70-complement-fixation-ELISA (Mo-rHSP70-CF-ELISA) and Mo-rHSP70-indirect-ELISA (Mo-rHSP70-iELISA). Both methods showed high sensitivity and specificity for the detection of M. ovipneumoniae antibodies in infected animal sera. A comparison between the two previously mentioned ELISA methods and Mo-iELISA (an indirect ELISA method which uses M. ovipneumoniae strain Y98 whole cell proteins as the coating antigen) was performed using 361 sheep clinical sera samples. The Kappa agreement coefficients between Mo-rHSP70-CF-ELISA and Mo-rHSP70-iELISA, Mo-rHSP70-CF-ELISA and Mo-iELISA, Mo-rHSP70-iELISA and Mo-iELISA, were 0.7640,0.8150 and 0.8649, respectively. Our data suggests that the assays established could be applied to diagnosis and epidemiological investigation of M. ovipneumoniae infection in sheep.In conclusion, EF-Tu and HSP70 of M. ovipneumoinae were able to induce the strong humoral immune response and cellular immune response. Both of EF-Tu and HSP70 might be good candidates for the development of M. ovipneumoniae subunit vaccine. The established Mo-rHSP70-CF-ELISA and Mo-rHSP70-iELISA maybe suitable for use as the methods of antibody detection in M. ovipneumoniae. |
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